New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Abstract

pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pH_in) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH_in for three prototrophic wild‐types. The new construct was also used to determine the pH_in in strains differing in the activity of the plasma membrane Pma1 H⁺‐ATPase and the influence of glucose on pH_in. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands‐on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright © 2010 John Wiley & Sons, Ltd.